A fundamental mechanism by which opioid receptors are regulated is by rapid endocytosis after agonistinduced activation. Receptors traverse divergent intracellular membrane pathways after their initial endocytosis, and receptor "sorting" between distinct downstream pathways plays a critical role in determining the functional consequences of endocytosis. The proposed studies seek to elucidate mechanisms that determine whether endocytosed opioid receptors recycle rapidly to the cell surface (a well characterized pathway promoting functional recovery or "resensitization" of signal transduction), or if they traffic to lysosomes (a well established pathway of proteolytic "down-regulation" that leads to a prolonged attenuation of signal transduction). The Specific Aims of the proposed studies are to: (1) Define the biochemical properties of a mechanism that mediates signal-dependent recycling of opioid receptors; (2) Elucidate a distinct mechanism that promotes sorting of endocytosed opioid receptors to lysosomes; (3) Determine whether opioid receptors are sorted between distinct membrane domains of multivesicular endosomes; and (4) Investigate the functional relevance of specific post-endocytic sorting mechanisms to opioid receptor regulation in neurons. These studies have general relevance to understanding mechanisms by which G protein-coupled receptors are regulated and specific relevance to mechanisms underlying physiological adaptation of the nervous system in response to clinically important opiate drugs.